Membrane-bound receptor activator of NFκB ligand (RANKL) activity displayed by osteoblasts is differentially regulated by osteolytic factors

Osteoclast formation is central to bone metabolism, occurring when myelomonocytic progenitors are stimulated by membrane-bound receptor activator of NFκB ligand (RANKL) on osteoblasts. Osteolytic hormones induce osteoblast RANKL expression, and reduce production of RANKL decoy receptor osteoprotegerin (OPG). However, rather than RANKL and OPG mRNA or protein levels, to measure hormonally-induced osteoclastogenic stimuli the net RANKL activity at the osteoblast surface needs to be determined. To estimate this we developed a cell reporter approach employing pre-osteoclast RAW264.7 cells transfected with luciferase reporter constructs controlled by NFκB (NFκB-RAW) or NFATc1 (NFAT-RAW)-binding promoter elements. Strong signals were induced in these cells by recombinant RANKL over 24 h. When NFκB-RAW cells were co-cultured on osteoblastic cells (primary osteoblasts or Kusa O cells) stimulated by osteolytic factors 1,25(OH)2 vitamin D3 (1,25(OH)2D3) and prostaglandin E2 (PGE2), a strong dose dependent signal in NFκB-RAW cells was induced. These signals were completely blocked by soluble recombinant RANKL receptor, RANK.Fc. This osteoblastic RANKL activity was sustained for 3 days in Kusa O cells; with 1,25(OH)2D3 withdrawal, RANKL-induced signal was still detectable 24 h later. However, conditioned medium from stimulated osteoblasts induced no signal. TGFβ treatment inhibited osteoclast formation supported by 1,25(OH)2D3-treated Kusa O cells, and likewise blocked RANKL-dependent signals in NFAT-RAW co-cultured with these cells. These data indicate net RANKL stimulus at the osteoblast surface is increased by 1,25(OH)2D3 and PGE2, and suppressed by TGFβ, in line with their effects on RANKL mRNA levels. These results demonstrate the utility of this simple co-culture-based reporter assay for osteoblast RANKL activity.

► RANKL activity in osteoblasts is of great interest but difficult to measure.
► The study demonstrates a co-culture method to estimate functional RANKL stimulus.
► The study shows that mRNA level of RANKL reflects the net functional RANKL.
► The study demonstrates that induced RANKL activity is sustained over 3 days.
► The approach applied provides a useful and rapid method to study RANKL activity.